CL01096070s

RESEARCHOpenAccessSalmonellaTyphimurium-specificbacteriophageFSH19andtheoriginsofspeciesspecificityintheVi01-likephagefamilyStevenPTHooton1,AndrewRTimms1,JoannaRowsell2,RayWilson2andIanFConnerton1*AbstractBackground:Wholegenomesequencingofbacteriophagessuitableforbiocontrolofpathogensinfoodproductsisapre-requisitetoanyphage-basedinterventionprocedure.
TrialsinvolvingthebiosanitizationofSalmonellaTyphimuriuminthepigproductionenvironmentidentifiedonesuchcandidate,FSH19.
Results:Thisphagewassequencedandanalysisofits157,785bpcirculardsDNAgenomerevealedanumberofinterestingfeatures.
FSH19constitutesanothermemberoftherecently-proposedMyoviridaeVi01-likefamilyofphages,containingS.
Typhi-specificVi01andShigella-specificSboM-AG3.
AtthenucleotidelevelFSH19ishighlysimilartophageVi01(80-98%pairwiseidentityoverthelengthofthegenome),withthemajordifferenceslyingintheregionassociatedwithhost-rangedetermination.
AnalysesoftheproteinsencodedwithinthisregionbyFSH19revealedaclusterofthreeputativetailspikes.
Ofthethreetailspikes,twohaveproteindomainsassociatedwiththepectatelyasefamilyofproteins(Tsp2)andP22tailspikefamily(Tsp3)withtheprospectthattheseenableSalmonellaOantigendegradation.
TailspikeproteinsofVi01andSboM-AG3arepredictedtocontainconservedright-handedparallelb-helicalstructuresbuttheinternalproteindomainsarevariedallowingdifferenthostspecificities.
Conclusions:TheadditionorexchangeoftailspikeproteinmodulesisamajorcontributortohostrangedeterminationintheVi01-likephagefamily.
Keywords:Phagebiocontrol,biosanitization,bacteriophagegenomics,SalmonellaTyphimurium,Myoviridae,P22-liketailspike,pectatelyasetailspikedomain,lipopolysaccharideBackgroundTheuseofvirulentbacteriophages(phages)asbiologicalcontrol(biocontrol)agentsagainstbacterialpathogensisanexpandingfieldofresearchaimedatproducingsus-tainablesolutionsforthecontrolofthesepathogens,andtocircumventproblemssuchasthoseassociatedwiththedevelopmentofmultidrug-resistantbacteria[1].
Theantimicrobialactivitiesofphagescommittedtothecellularlysisofarangeofbacterialpathogenshavebeenreported,whichincludefoodpathogenssuchasCampy-lobacterjejuni[2-5],Escherichiacoli[6-8],andvariousSalmonellaentericaserovars[4,9,10].
However,despitethewealthofdataregardingtheefficacyofphagesdur-inginterventionstudies,relativelyfewphage-derivedproductshavebeendevelopedsufficientlyforcommer-cialapplication.
Onlyinthelastfewyearshastherebeenanextensionoflab-basedtrialsintothefoodpro-ductionenvironment,whereperhapsthebestexampleistherecognitionoftheefficacyandthegrantingof'gen-erallyrecognizedassafe'(GRAS)statustobacterio-phagestargetingListeriamonocytogenesbytheUnitedStatesFoodandDrugAdministration[11].
Therearetwoproductsofnotenowavailablecommercially-List-Shield(IntralytixInc.
,USA)aphagecocktailcompris-ingvirulentphageswithbroadactivityagainstL.
Monocytogenes,andListexP100(EBIFoodSafety,Netherlands).
FollowingGRASclassificationbothList-ShieldandListexP100arenowviewedassafeto*Correspondence:ian.
connerton@nottingham.
ac.
uk1DivisionofFoodSciences,SchoolofBiosciences,UniversityofNottingham,SuttonBoningtonCampus,LoughboroughLE125RDUKFulllistofauthorinformationisavailableattheendofthearticleHootonetal.
VirologyJournal2011,8:498http://www.
virologyj.
com/content/8/1/4982011Hootonetal;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
beappliedasfoodbiopreservativesonready-to-eatfoodsintheUSA.
PhageP100(theactivecomponentofListexP100)wasinitiallycharacterizedatthegeneticlevelandinoraltoxicitystudiesbyCarltonetal[12].
Thesestu-diesshowedthatP100hadnoundesirablegeneswithinitsgenome,andcausednoilleffectswhenadministeredtorats.
ManystudiesreportingtheefficacyofListexP100againstL.
monocytogenesinvariousfoodproduc-tionsettingsarenowavailableintheliterature[13-15].
Ifmorephage-basedapplicationsaretoreachstandardswheretheyaredeemedfitforhuman/animalconsumptionthencertainmatterspertainingtosafetymustbetakenintoconsideration.
Forinstance,phagesthatshowpoten-tialduringpreliminarystudiesmustadheretostrictcri-teriaiftheyaretobedevelopedfurtherasantimicrobialagents[16].
Mostimportantly,theselectedphagesmustnotpossessgenesassociatedwithvirulence,orthosethatmayenhancethepathogenicprofileofitstarget[17].
Manyexamplesofphagesthatarerecognizedasbeinginvolvedinsuchprocessesareknown,forexample,theShigatoxin-encoding(Stx)phages-akeyvirulencefactorofShiga-toxigenicE.
coli(STEC)-arethecausativeagentsofhaemolyticuraemicsyndrome(HUS),amajorcontribu-tortodiseaseassociatedwithSTECinfection[18,19].
Also,temperatephagesthathavethepotentialtoformlysogenswiththeirhostneedtobeeliminatedfromtrialsattheearliestopportunity.
Integrationinto,andexcisionoutofthehostgenome,canleadtothetransferofgenesbetweenthephageandbacteriumpotentiallyalteringthegeneticprofileofboth[20].
TheseundesirabletraitsaremostoftenassociatedwithphagesbelongingtothePodoviridae(icosahedralheadwithashortnon-contractiletaile.
g.
Sal-monellaphageP22)orSiphoviridae(icosahedralheadwithaflexiblenon-contractiletaile.
g.
phagel)families.
However,membersoftheMyoviridae(icosahedralheadandcontractiletaile.
g.
phageT4)aremoreoftenasso-ciatedwithanobligatelylyticlifecycle.
Allofthesemor-phologicalcharacteristicscanbeeasilydiscernedwhenphagesareviewedunderatransmissionelectronmicro-scope,althoughassumptionsastothegeneticnatureofaphagecannotbemadeonmorphologicalcharacteristicsalone.
Wholegenomesequencingofphageisolatesintendedforuseasbiocontrolagentsisnowconsideredtobethe'goldstandard'intermsoftransferringphage-basedapplicationsfromthelaboratorytoeverydayuse.
HerewereportthecompletegenomesequenceofFSH19,alyticbacteriophageadaptedtoinfectinganumberofdifferentS.
Typhimuriumserovars.
FSH19hasshowngreatpotentialasabiocontrolagentagainstS.
TyphimuriumU288,themostprevalentserovarfoundinUKpigproductionpremises[21].
S.
TyphiphageVi01wasthefirstofarecently-proposednewlineageofMyoviridaetobedescribed.
GeneticanalysesoftheFSH19genomerevealthatitisacloserelativeofphageVi01,intermsofbothDNAandproteinsequences.
However,phageVi01appearstoberestrictedtoinfectingS.
Typhi,possiblyduetothepresenceofavirulence(Vi)capsuleantigen-degradingacetylesterasedomainincorporatedintooneofitsthreetailspikes[22].
TheothercompletelysequencedmemberoftheVi01-likephagelineageisSboM-AG3thatisrestrictedtotheinfectionofShigellaspp.
[23].
Nogenesasso-ciatedwitheithertoxicityorlysogenyhavebeenfoundwithintheFSH19157,785bpcirculardsDNAgenome,norinanyoftheVi01familyofphages.
Alackofviru-lenceassociatedgenesinFSH19willallowitsuseasabiocontrolagent,aimedatreducingS.
Typhimuriumenteringthefoodchain,andinparticularS.
Typhimur-iumU288fromporkproduction.
ResultsBacteriophageFSH19characterizationBacteriophageFSH19wasoriginallyisolatedfrompigintestinalcontentsandhasspecificactivityagainstS.
Typhimuriumserovars[24].
MorphologicallythephagehasanicosahedralheadwithacontractiletailindicatingittobeamemberoftheMyoviridae(Figure1).
Acom-parisonofthelyticprofilesofphagesFSH19andVi01againstapanelofS.
entericaSerovarsisgiveninTable1.
ThedatashowsthatasexpectedalltheS.
Typhimur-iumstrainstestedarerefractorytoinfectionbyVi01,whereasS.
TyphiBRD948likewisedisplaysimmunitytowardsinfectionbyFSH19butsusceptibilitytoVi01.
GenomeanalysisTheFSH19genomewassequencedfromsonicatedDNAfragmentsusingtheRoche454GSFLXplatformFigure1TEMimageofFSH19.
ThepresenceofanicosahedralheadandcontractiletailindicatethatFSH19isamemberoftheMyoviridae.
Magnification*43,000.
Bar250nm.
Hootonetal.
VirologyJournal2011,8:498http://www.
virologyj.
com/content/8/1/498Page2of14(17,796reads),fromwhichasinglecontigof157,785bpwasgenerated.
BlastNanalysisoftheFSH19genomerevealedtworelatedgenomesequencesinthedatabase-phagesVi01(GenbankAcc.
No.
FQ312032)andSboM-AG3(GenbankAcc.
No.
FJ373894).
TheFSH19gen-omewasorderedsuchthatitcouldbedirectlyalignedwiththeexistinggenomesofVi01andSboM-AG3com-mencingwiththerIIAgene.
TheGC-contentoftheFSH19genomewasdeterminedtobe44.
68%,whichisslightlylowerthanVi01(45.
22%GC),andconsiderablylowerthanSboM-AG3(50.
39%GC).
AlignmentofthenucleotidesequencesofFSH19andVi01indicatethattheyarehighlysimilaroveralargeproportionofthegenome(between80-98%pairwiseidentity).
However,foraregionlocatedbetween120-130Kbonthegenomemapthishomologybreaksdowntoapproximately70%identity(andinsomeinstancestheconservationislostcompletely).
ThisregioncontainsaclusterofthreeputativetailspikereadingframesinalloftheVi01-likephagessequencedtodate.
AtthenucleotidelevelFSH19issomewhatlesssimilarwhencomparedtoSboM-AG3,withhomologiesrangingfrom74-100%identityoverapproximately60%ofthegenome.
Com-parisonofthevaryingdegreesofhomologybetweenFSH19,Vi01,andSboM-AG3arerepresentedinFigure2.
OtherwisehighlysimilarnucleotidematcheswererestrictedtocomponentsofEscherichiaphagePhaxI(GenbankAcc.
No.
HQ259289),SerratiaphageKSP90(GenbankAcc.
No.
AB452990)andSalmonellaphageDet7(GenbankAcc.
No.
AM765843).
InitialanalysisoftheFSH19genomeidentified339potentialopenreadingframes(ORFs).
FollowingBlastPandPfamdomainanalysesofeachpotentialORF,thecandidateORFswerereducedto166.
EachORFwasthenannotatedusingVi01andSboM-AG3phagesasreferencesequences,andcategorisedasfollows:Hypotheticalphageproteins(81),Conservedhypotheti-calphageproteins(23),DNAreplication(24),Tailmor-phogenesis(16),Capsid/DNApackaging(7),DNAmaintenance/circularization(6),Putativeuncharacter-izedproteins(3),Regulators(2),Lysis(2),andPutativehomingendonucleases(2).
AcomparisonofthemajorproteinsofFSH19withtheirhomologuesinVi01andSboM-AG3ispresentedinTable2.
Figure3showsthefullyannotatedFSH19genomeasacirculargeneticmap.
UsingtRNAscan-SE,fivetRNAgeneswerealsoidentifiedina5Kbnon-codingregionoftheFSH19genomeatthefollowingnucleotidepositions:Methio-ninetRNA(CAT)137,703-137,735;AsparaginetRNA(GTT)137,806-137,878;TyrosinetRNA(GTA)138,497-138,577;SerinetRNA(GCT)138,657-138,742;andanundeterminedtRNA138,749-138,834.
ForphagesFSH19,Vi01,andSboM-AG3thereexistsasetoftRNAgeneslocatedatsimilarpositionsintheirrespec-tivegenomes,someofwhichdisplayahighdegreeofconservation.
Forexample,BlastNanalysisofthecodingsequencefortRNA-Serinethatispresentinallthreephagesindicates100%identityatthenucleotidelevel.
TwoothertRNAs(tRNA-AsparagineandtRNA-Tyro-sine)arealsosharedbetweenthegroup.
ForFSH19tRNA-Asparagine,thegeneis100%identicalwithitshomologueinVi01and95%identicaltothatofSboM-AG3,whilsttheFSH19tRNA-TyrosineshowsmoreconservationwiththatofSboM-AG3(99%identical)thanVi01(84%identical).
FSH19alsosharesatRNA-Methioninegene(88%identity)andatRNAofundeter-minedspecificity(99%identity)withVi01thatareabsentinSboM-AG3.
Phireanalysisforphageregula-toryelementsidentifiedanumberofsitesintheFSH19genome(Table3).
Alloftheputativeregulatoryele-mentsarefoundonthenon-codingstrand(relativetothetranscribedsequenceswithinthatregion)howevertheyappeartobeassociatedwithORFsontheoppositestrand.
ItisquitepossiblethattheseelementsplayaroleinregulatingFSH19geneexpression,inanasyetundeterminedmanner,butperhapsbytheproductionofsmallRNAmolecules.
FSH19tailspikesThreetailspikegenes-tsp1(bases120,710-122,641c),tsp2(122,702-124,876c),andtsp3(124,992-127,088c)wereidentifiedduringtheannotationofFSH19.
BlastNanalysisofthetsp1gene(1932bp)showsthatthenucleotidesequencehas94%identitywiththatofVi01orf170c(Vi01Tsp1)overthefirst263bases,butbeyondthisconservationbreaksdownandnofurthernucleotideidentitiesareobservedbetweenthetwogenes.
Asimilarsequencetotsp1ispresentatorf00207(SboM-AG3Tsp1)ofSboM-AG3(64%identityoverthefirst390bases),andashorterstretchofhomology(78%identityoverthefirst54bases)islocatedwithinorf00212(SboM-AG3Tsp3).
AswithVi01,nofurtheridentitycanbeobservedwithtsp1followingtheseshortstretchesofconservation.
Othernucleotidealignmentsoftsp1includeidentitieswiththeSalmonellaphageDet7tailspikegene(86%identityfrombases360-460),Table1HostrangeassaysofFSH19andVi01(+=lysisand-=nolysis).
SalmonellaFSH19Vi01S.
TyphimuriumU288+-S.
TyphimuriumDT104WT+-S.
TyphimuriumDT104NCTC13348--S.
TyphimuriumWT(Rawlings)+-S.
TyphimuriumWT(Turner)+-S.
TyphimuriumLT2+-S.
TyphiBRD948-+Hootonetal.
VirologyJournal2011,8:498http://www.
virologyj.
com/content/8/1/498Page3of14andanendo-a-sialidasegeneofcoliphageK1F(76%identicalfrombases351-485).
Asidefromthesealign-ments,whichallspanaregioncoveringthefirst485basesoftsp1,nohomologieswerefoundinthedatabasefortheregioncoveringbases485-1932.
Alignmentofthetsp2gene(2175bp)revealsthatonlyVi01andSboM-AG3havesimilarsequenceswithinthedatabase.
Aswithtsp1,thehomologiesallspanthestartofthegene:85%identity(bases1-723)withVi01orf171c(Vi01Tsp2-maturation/adhesionprotein)and70%iden-tity(bases1-360)withSboM-AG3orf00210(SboM-AG3Tsp2).
Thelatterportionofthetsp2genedoesnotalignwithanysequencesinthedatabase.
BlastNsearchesinvolvingthetsp3gene(2097bp)showthatthesequenceishighlysimilartotheDet7tailspikegene(85%identityfrombases1-782).
Toalesserextent,tsp3sharessequencehomology(84%identitybetweenbases1-461)withVi01orf172c(Vi01Tsp3)andorf170c(69%identitybetweenbases1-179).
Apartfromtheseshortregionsofhomology,therearenofurtheridenti-tiesbetweentsp3andVi01.
Interestingly,nonucleotidesequencehomologywasfoundbetweentsp3andSboM-AG3.
Bases481-667showaconservedsequencethatissharedbetweenseveralphagegenomesinthedatabase.
Sequencealignmentsoverthisregionindicate74-77%sharedidentitiesbetweenvariousSalmonellaphagesincludingP22,ST104,ST64T,ST160,SE1,andsomeoftheS.
Enteritidistypingphages(SETP)[25].
Interest-ingly,thesequenceisalsopresentinprophagetailspikesofS.
TyphimuriumD23580,S.
TyphimuriumT000240,S.
HeidelbergSL476,andS.
ParatyphiA(strainAKU12601).
Aswasthecasefortsp1andtsp2nohomologoussequencesarefoundtomatchthelatterregionofthetsp3gene.
TranslationofthetspgenesequencesindicatethatTsp1isa643aminoacidprotein(predictedMW~68.
9kDa),Tsp2iscomprisedof724aminoacids(predictedMW~78.
2kDa),andTsp3698aminoacids(predictedMW~75.
8kDa).
PfamdomainsearchesforeachtailspikeindicatedsignificantdomainmatchesforTsp2(Pectatelyasedomain-family3CL0268)andTsp3(P22tailspikefamily);howevernosignificantdomainFigure2ArtemisComparisonTool(ACT)analysisofthegenomesofSboM-AG3(top),FSH19(middle),andVi01(bottom).
Hootonetal.
VirologyJournal2011,8:498http://www.
virologyj.
com/content/8/1/498Page4of14Table2ComparisonofFSH19proteinswiththoseofVi01andSboM-AG3CDSFSH19coordinates&aminoacidlengthPredictedMWPfamdomainAminoacidIDwithVi01AminoacidIDwithSboM-AG3RIIA1-2742(913)104,992None98%54%RIIB2775-4358(527)58,980None97%69%Putativetailfibre5502-6311(269)27,826Ig-likeI-setdomain(CL0011)86%86%DNAtopoisomeraseII8523-10,436(637)71,862HATPasec(CL0025)&DNAgyraseB96%90%DNAtopoisomeraseII(mediumsubunit)10,357-11,769(470)53,939DNAtopoisomeraseIV88%88%Conserveduncharacterizedprotein13,309-13,929(206)23,005PutativeMacrodomainofADP-ribosebindingmodule(CL0233)92%74%DexAexonuclease14,263-14,895(210)24,438None95%93%dCMPdeaminase18,704-19,225(173)19,460dCMPcytosinedeaminase1family-cytidine&deoxycytidylatedeaminaseZn-bindingdomain(CL0109)97%62%Conserveduncharacterizedprotein19,227-19,634(135)15,378Bacterialmembrane-flankeddomain(DUF304)93%88%Headcompletionprotein19,845-20,465c(206)24,189None92%74%Putativehomingendonuclease20,465-21,205c(246)28,205GIY-YIGcatalyticdomain(CL0418)Nomatch34%T4-likebaseplatetailtubecap21,259-22,227(322)36,142T4tailcapfamily100%94%Baseplatewedgesubunit22,240-22,797(185)21,574PhageGp53family97%80%LoaderofT4-likehelicase26,134-26,796c(220)26,253T4helicaseNfamily99%79%DNAligase27,281-28,708c(475)53,501ATP-dependentDNAligasedomain(CL0078)95%83%DNAprimase-helicasesubunit31,451-32,875c(474)54,192DnaB-likehelicaseN&Cterminaldomains98%88%UvsX(RecA-likeprotein)33,189-34,274c(361)40,791RecAbacterialDNArecombinationproteins(CL0023)72%78%dUTPase34,789-35,340c(183)21,114dUTPase2family(CL0231)69%63%Thymidylatesynthase35,903-36,943c(346)39,216Thymidylatesynthasefamily93%77%DNAendprotector39,924-40,268c(234)28,347None100%91%Baseplatetailtube40,488-41,624(378)42,658PhageT4Gp19family98%90%ssDNAbindingprotein41,652-42,683c(343)38,896Gp3DNAbindingprotein-likedomain98%77%Latepromotertranscriptionfactor43,029-43,331c(100)11,175None100%70%Regulatoryprotein(FmdBfamily)43,264-43,509c(81)8732CXXCCXXCSSSSfamilycontainingaZnribbondomain(CL0167)96%88%Putativeuncharacterizedprotein43,875-44,396c(203)23,739RuvCfamily(crossoverjunctionendoribonucleaseRuvC)100%83%Baseplatehubsubunit45,471-46,274(267)30,186T4baseplatefamily96%67%Tail-associatedlysozyme46,781-48,406(541)59,023Gp5OBfamily&CHAPdomain(CL0125)associatedwithpeptidoglycanhydrolysis98%87%Baseplatewedgeprotein48,471-48,851(126)14,124GPWGp25family(gene25-likelysozyme)97%87%NrdB50,229-51,332c(367)42,209Ribonucleotidereductasesmallchaindomain(CL0044)99%89%NrdA51,403-53,730c(775)88,137ATP-conedomain,ribonucleotidereductaseIgNallalphadomain&IgCbarreldomain94%89%PhoH-likephosphatestarvation-inducibleprotein53,764-54,603c(279)31,567PhoH-likeproteinfamily(CL0023)100%88%Peptidoglycanbindingprotein54,711-55,505c(264)28,815Peptidoglycanbindingproteindomain(CL0244)&proteinofunknownfunction(DUF3380)98%86%DNAprimasesubunit56,712-57,776c(354)41,386None97%77%Conserveduncharacterizedprotein63,656-64,273c(205)22,902T4RegBendoribonucleasefamily(CL0037)98%79%Hootonetal.
VirologyJournal2011,8:498http://www.
virologyj.
com/content/8/1/498Page5of14Table2ComparisonofFΦSH19proteinswiththoseofVi01andSboM-AG3(Continued)NrdA.
164,978-65,313c(336)12,934None94%92%Recombinationendonucleasesubunit65,622-67,955c(777)88,531None98%72%Recombinationproteinsubunit67,958-69,073c(371)43,030None98%91%sfactorforlatetranscription69,060-69,854c(264)30,775None90%77%Putativehomingendonuclease69,854-70,576c(240)27,188NoneNomatch36%RNaseH70,587-71,114c(175)19,878RNaseHdomain(CL0219)97%71%ATP-dependenthelicase71,921-73,522c(533)60,690None99%86%DNAbindingprotein73,778-74,056c(92)9728BacterialDNA-bindingproteindomain100%86%Conserveduncharacterizedprotein74,140-74,931c(263)28,556SPFHdomain/Band7family(CL0433)98%NomatchSuperinfectionexclusionprotein75,137-75,457c(106)12,562None95%NomatchImpD75,736-76,407c(223)25,665None95%74%Acylcarrierprotein80,335-80,687c(110)12,162Phosphoantetheine(PP)-bindingfamily(CL0314)92%61%Conserveduncharacterizedprotein84,506-84,925c(139)15,622Proteinofunknownfunction(DUF3268)90%84%PutativeRegAtranslationalrepressor89,926-90,390c(154)18,078Bacteriophagetranslationalregulator99%90%ClamploaderforDNApolymerase90,420-90,842c(140)16,200None98%76%Gp44slidingclampholder90,847-91,836c(329)37,214ATPasefamily(CL0023)99%87%Gp45slidingclampholder91,914-92,582c(222)24,510Gp45slidingclampCterminal99%82%PutativetypeIIIrestrictionenzyme(RE)93,293-94,792(499)57,802TypeIIIREsubunit(CL0023)&helicaseconservedCterminaldomain(CL0023)95%81%Conserveduncharacterizedprotein94,822-95,568c(248)28,911PD-(D/E)XKnucleasesuperfamily97%86%PutativeUvsY95,568-96,023c(151)17,991UvsYproteinfamily(recombination,repair,&ssDNAbindingprotein93%85%Tailcompletionprotein96,062-96,559c(165)18,542T4Gp19family98%80%Majorcapsidprotein101,588-102,910c(440)48,059Gp23majorcapsidproteinfamily98%94%Proheadcorescaffoldprotein103,002-103,841c(279)30,898None96%74%Proheadprotease103,888-104,556c(222)24,482PeptidaseU9family(CL0201)99%92%Portalvertexproteinofthehead105,102-106,784c(560)63,094T4Gp20family99%81%Tailtubeprotein106,852-107,385c(177)19,912T4Gp19family100%98%GIY-YIGendonuclease107,416-107,874c(152)17,316GIY-YIGcatalyticdomainendonucleasefamily(CL0418)95%32%Tailsheathprotein107,933-109,828c(631)68,439Phagesheath1family98%92%Largeterminasesubunit109,881-112,091c(736)84,547Terminase6family95%85%Smallterminasesubunit112,072-112,752c(226)24,825DNApackagingfamily(terminaseDNApackagingenzyme)98%74%Proximaltailsheathstabilization112,755-113,453c(232)26,979None98%80%Gp14neckprotein113,456-114,097c(213)24,759T4neckproteinfamily100%82%Gp13neckprotein114,400-115,209c(269)31,142None99%87%Hootonetal.
VirologyJournal2011,8:498http://www.
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com/content/8/1/498Page6of14similaritieswereidentifiedforTsp1.
BetawrapanalysesoftheaminoacidsequencesofeachtailspikefromFSH19,Vi01,andSboM-AG3showsthateachoftheseproteinspotentiallyhavetheabilitytoformright-handedparallelb-helicalstructures.
AcomparisonofeachFSH19tailspikeproteinsequencewiththoseofVi01andSboM-AG3,andotherhomologousproteinsinthedatabaserevealedtheN-terminalresidues(1-130)ofFSH19Tsp1arehighlyconservedwiththecorrespond-ingregionsinVi01Tsp1(78%ID),SboM-AG3Tsp1(75%ID),andtoalesserdegreeinVi01Tsp3(56%ID).
ThisN-terminalhomologyisalsosharedwiththeTable2ComparisonofFΦSH19proteinswiththoseofVi01andSboM-AG3(Continued)Tailspike1120,710-122,641c(643)68,851None67%(res.
1-171only)62%(res.
1-190only)Tailspike2122,702-124,876c(724)78,194PectatelyaseC(CL0268)86%(res.
1-276only)73%(res.
1-161only)Tailspike3124,992-127,088c(698)75,785P22tailspikefamily86%(res.
1-154only)46%(res.
1-179only)Haemolysin-typecalciumbindingprotein127,168-130,209c(1013)108,436None81%(res.
1-418only)63%(res.
1-404only)Baseplatewedgesubunit132,287-134,080c(597)66,068None98%79%DNApolymerase145,442-148,438(998)116,583DNApolymerasefamilyBexonucleasedomains(CL0194&CL0219)98%86%Putativeuncharacterizedprotein148,776-149,615(279)32,346NT5Cfamily5'nucleotidasedeoxypyrimidine(CL0137)97%80%Figure3AschematiccircularmapoftheFSH19genome.
Hootonetal.
VirologyJournal2011,8:498http://www.
virologyj.
com/content/8/1/498Page7of14SalmonellaphageDet7tailspike(56%ID),andatailfibreproteinfromEnterobacterphageEcp1(42%ID).
AsequencemotifpresentinFSH19Tsp1G-G-V-G-L-G-A-W(beginningatresidue143)possiblysignifiestheboundaryatwhichthecatalyticdomainofthetailspikebegins.
NosuchsequencemotifispresentinVi01Tsp1,howeversimilarsequencemotifsarepresentatanalo-gouspositionswithinSboM-AG3Tsp1(G-G-L-S-S-S-N-W)andVi01Tsp3(G-G-V-G-T-G-A-W).
ClustalW2alignmentsofphagetail-associatedproteinscontainingderivativesofthesemotifsattheirN-terminalbound-ariesareshowninFigure4A.
TheC-terminusofFSH19Tsp1(residues300-643)producednosignificantalignmentswithanyknownproteins.
BlastPanalysesofVi01Tsp1andSboM-AG3Tsp1wasperformedtoiden-tifyanyfunctionally-relatedproteins.
However,aswithFSH19Tsp1,Vi01Tsp1producednosignificantalign-mentsovertheC-terminalregionwithanyproteinsequencesinthedatabase.
AlignmentofFSH19Tsp2withVi01Tsp2(matura-tion/adhesionprotein)andSboM-AG3Tsp2showsahighdegreeofconservationfromresidues1-160.
AtthispointSboM-AG3sequencehomology(73%IDoverresi-dues1-162)breaksdownwithnofurtheridentitytoeitherVi01orFSH19Tsp2.
Residues100-403inFSH19Tsp2showaweakbutsignificantrelationshipwithaphagestructuralprotein/putativetailfibre(orf00213)presentinSboM-AG3.
ForFSH19Tsp2andVi01Tsp2,homologycontinuesoverthefirst276residues(86%ID)atwhichpointthetwosequencescompletelydivergefromeachother.
Anotherregionoflowhomology(resi-dues100-316)isalsofoundwithaputativetailfibrepro-teinencodedintheVi01genome(Vi01orf173c).
Apectatelyaseproteinfamilydomain(family3-CL0268)spanningresidues291-549wasidentifiedduringPfamanalysisofFSH19Tsp2.
ThepresenceofthismotifwasfurtherconfirmedduringBlastPanalysis,withanumberofalignmentsbeingmadewithvariousglycosidedegrad-ingenzymesfromawiderangeofmicroorganisms.
NosignificantalignmentswiththeC-terminusofFSH19Tsp2wereidentified(residues557-724).
FSH19Tsp3alignmentwithVi01Tsp3showsahighdegreeofsequenceconservationbetweentheirN-term-inalresidues1-154(86%ID).
However,thecorrespond-ingsequenceinSboM-AG3Tsp3showssimilarityoverthefirst19residuesbeforeadeletionof63aminoacids,afterwhichthehomologyisrestoredbetweenthethreetailspikesequences.
Interestingly,SboM-AG3Tsp1showsahigherdegreeofconservationwithFSH19Tsp3overresidues1-139(46%ID)thandoesSboM-AG3Tsp3.
TheboundarymotifG-G-V-G-L-G-A-WappearsinFSH19Tsp3atresidues143-150withanalo-goussequencesinVi01(G-G-V-G-T-G-A-W)andSboM-AG3(G-G-V-S-S-S-A-W),afterwhichthesequencehomologiesbetweenallthreetailspikesbreakdown.
PfamdomainanalysisofFSH19Tsp3identifiedaproteindomain(residues159-698)withsignificanthomologytotheP22tailspikefamily(PF09251).
BlastPanalysisproducedalignmentswithDet7(thefirstreportedMyoviruswithapodoviraltailspike),P22,andanumberofP22-likephages.
ThehighestdegreeofhomologywasfoundforthetailspikeofDet7(77%IDoverthefirst300residues).
ThesequencemotifmarkingthedomainboundaryinFSH19Tsp3(G-G-V-G-L-G-A-W)isconservedinDet7.
P22tailspike-likesequencesfeaturethreeconservedresiduesthatfunctionascataly-ticcomponentsoftheendo-rhamnosidaseactivityasso-ciatedwithP22tailspikeprotein[26,27].
TheseresiduesarelocatedinthesubstratebindingcleftoftheP22tailspikeandareconservedinFSH19Tsp3(Figure4B:Glu-359,Asp-392andAsp-395).
DiscussionBacteriophageFSH19,acandidateforuseasabiocon-trolagentagainstSalmonellaTyphimurium,wassub-jectedtowholegenomesequencing.
Thisprocessisnowviewedasapre-requisitetousingphagesastherapeuticagents,especiallyiftheyaretobeintroducedintoafoodproductionenvironment.
Thepresenceorabsenceofgenesassociatedwithtoxicityorlysogenywillulti-matelygovernwhetherornotapotentialphagecandi-dateissafeforcommercialuse.
FSH19wasfoundtocontainnoundesirablegenesinits157,785bpgenomemakingitanexcellentcandidateforsuchapplications.
Also,FSH19constitutesanewadditiontotheVi01-likephagefamily(alongwithS.
TyphiphageVi01andShi-gellaphageSboM-AG3).
InmanyrespectsFSH19isquitesimilartoVi01withtheadditionofaputativehomingendonucleaseandaputativeuncharacterizedprotein.
Structuralgenesandthoseassociatedwithmor-phologyofthephageparticlearehighlyconservedbetweenFSH19andVi01,andtoalesserdegreewithSboM-AG3.
AnalysisofthetRNAgenesencodedbyallthreephages,andtheirlocationwithineachgenomeindicatesthatsomeofthemarepossiblyderivedfromaTable3PutativephageregulatoryelementsidentifiedbyPhireanalysis.
PutativephageregulatoryelementsFromToSequence56,30056,319TAGCCATGATGTAATTCCTC79,92679,945CGGTCATGATGTATTTCCTT99,48799,506TGGTCATGGTGTAGTTCCTT101,431101,450GTCCCATGATGTATTTCTTC104,872104,891TTTCCATCATGCATCTCCTT109,822109,841TTGCCATAATATCATTCCTT144,434144,453TTGCCATGATGTATTTCCTTHootonetal.
VirologyJournal2011,8:498http://www.
virologyj.
com/content/8/1/498Page8of144ADet7TspPKVVPAGSTPASTGGVGLGAWVSVGDAAFRQEAN-KKFKYSVKLSDYS176SETP12TspPKVIPAASTPLTAGGISDSAWVAFGDITFRAEAD-KKFKYSVKLSDFT153Phi19Tsp3PKTVDAGSTPETSGGVGLGAWLSVGDAAFRQEAN-KKFKYSVKLSDYS176k1HTsp63DsialidasePKTIPASSSPQSTGGVSEGAWQRAGSVVLRQQISNANGAILYPDLHVARWKDEYDPRAWG166K1GTsp63DsialidasePKIIPASSSPQSTGGVADGAWQPVGSVILRQQISDVNGAILYPDLHVARWKDEYDPRAWG166K1FtailfibrePKNVPAGSTPETSGGIGLGAWVSVGDAALRSQISNPEGAILYPELHRARWLDEKDARGWG245Vi01Tsp3PKVVAAGSTPDNSGGVGTGAWIPVGINSLSGDGGWSRIGSEFGKSLKEHIQSLPSLSAVK189Phi19Tsp1PKTVAPGSTPATTGGVGLGAWVSVGDVSLRSELS-KEDGFKYIGYS172SboM-AG3orf00207PKSVNAGGTIANTGGLSSSNWVKLDPDALRDELY-SGFYRLT169EcoDS1gp17PKEVPAGSTPSTTGGVGLGAWVSVGDASLRSNLIS-DNSINGDALI230EPECa14tailfibrePKDVPENSTPESTGGVSLGAWVSVGDASLRSDLIS-QETDKGS293SboM-AG3orf00210PKTVNKNSTPQSTGGVSSSAWVSVESKTLGSELLTG-EKASLVG111K.
pneumoniaeprophagetailPKTVPIASSPATTGGVRAGGWAYTSDAAIRQNLAADSGAKLVGGLNYVTPEMSGFVTGAG152ViII-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ΦSH19Tsp3MGGTTNPSLLMTADCQVATPGGEASIVKLSAIQEGVRVGE641ST64TTspLKVNHKDCRGAEIPFVPDIASDDFIKDSSCFLPYWENNSTSLKALVKKPNGELVR661ST104TspLKVNHKDCRGAEIPFVPDIASDDFIKDSSCFLPYWENNSTSLKALVKKPNGELVR661P22TspLKVNHKDCRGAEIPFVPDIASDDFIKDSSCFLPYWENNSTSLKALVKKPNGELVR661SETP13TspLKINRGDYRAVEIPVAMSALPDNAVRDIGSISMYLEGD--SLKALVRRADGSYTR680SETP12TspLKINRGDYRAVEIPVAMTVLPDNAVRDNGSISLYLEGD--SLKALVKRADGSYTR680SP6TspLKINRGDFKATEIPVAPTVLPDEAVRDHSSIALYFDQE--ALWALVKKPNGSLTR546Det7TspLKVNRGDYKTTEIPISGTVLPDEGVLDINTMSLYLDAG--ALWALIRLPDGSKTR703ΦSH19Tsp3FQLNRLGFKHMSIPAAPLQLPESALEHNSSIGFFFGSDG-ALRLLAKKPDGSYVT695ST64TTspLTLATL---667ST104TspLTLATL---667P22TspLTLATL---667SETP13TspLTLA-----684SETP12TspLTLA-----684SP6TspMKLA-----550Det7TspMKLSV----708ΦSH19Tsp3YTL------698Figure4A-CClustalW2alignmentsofsequencemotifsintail-associatedproteins(4A),theactivesiteofP22-liketailspikes(4B),andtheC-terminalregionofP22-liketailspikes-yellow-sequencemotif(residues585-596inP22)associatedwithintertwinedregion,green-five-strandedb-sheetandthree-strandedb-sheet(residues601-664inP22),andred-regionslackinghomologyinFSH19Tsp3(4C).
Hootonetal.
VirologyJournal2011,8:498http://www.
virologyj.
com/content/8/1/498Page9of14commonancestor.
TransmissionelectronmicroscopeimagesofFSH19(Figure1),Vi01andSboM-AG3indi-catethattheirmorphologiesarequitesimilar.
Theintri-cate'chandelier-like'arrangementsoftailspikesattachedtothebaseplatearevisibleforallthreephages[22,23].
ItseemslikelythattherearemoreVi01-likephages(suchasEscherichiaphagePhaxI)capableofinfectingadiverserangeofbacterialpathogenstobediscoveredforwhichtherearecommercialapplications.
ThemodularstructureofthethreetailspikesfoundinFSH19ispotentiallythemaindrivingfactorbehindhost-rangedeterminationforthisphage.
AlthoughnofunctioncanatpresentbeassignedtoTsp1,theothertwotailspikescontaindefinedproteindomainsthatprovideevidenceastotheirmodeofaction.
ForTsp2,thepectatelyasedomainindicatesthatthisproteinmaywellfunctiontomodifyorcleaveglycosidebonds.
Thepectatelyasefamilydomain(CL0268)notablycontainsproteinswithpectate/pectinlyaseandpectinmethyles-teraseactivities.
Thepectate/pectinlyasesareawell-characterizedfamilyofproteinsprincipallyinvolvedinmicrobialplantpathogenesis.
Theirprimarymodeofactioninvolvestheeliminativecleavageofa-1,4linkedgalacturonosylresiduesofpectinsthatarecomponentsofthemiddlelamellaofplantcellwalls.
ThethreedimensionalstructureofErwiniachrysanthemiEC16pectatelyaseC(PelC)wasfirstsolvedbyYoderetal.
[28]andisrepresentativeofafamilyofproteinscon-tainingright-handedparallelb-helicalstructureswiththeflexibilitytoallowproteinloopsfromthestackedcoilstoaddfunctionality,forexampletheformationoftheactivesitecleftsthatsupportenzymecatalysis[29,30].
Usingasimilarbasearchitecturepectinmethy-lesteraseactstode-esterifypectintopectate.
Membersofthepectinolyticproteinfamilyarerepresentedinpro-karyoticandeukaryoticmicroorganisms,andalsofunc-tioninplantstoremodelcellwalls[29].
Divergencefromtheancestralsequenceovertimehasalloweddif-ferentmicroorganismstotargetarangeofpectin-likesubstrates,whiletheoverallstructurehasbeenmain-tained[30].
Itisoflittlesurprisethatphageshaveevolvedtheabilitytoacquiretheseproteinsandutilizetheirpolysaccharidemodifyingproperties.
InthecaseofFSH19Tsp2itcouldbepostulatedthatthetargetisthea-D-galactose(1-4)a-D-mannoselinkagefoundintheS.
TyphimuriumO-antigentrisacchariderepeat.
Manyphagetail-associatedproteinshavebeenfoundtocon-tainright-handedparallelb-helicalstructuraldomains,forexamplephagesP22[31],Sf6[32],HK620[33]andDet7[27].
Moreoverthepresenceofapectatelyase-typestructureinaphagetailspikeproteinhaspreviouslybeenreportedforK5lyase(KflA)ofcoliphagesK5AandK1-5.
KflAtargetsanddegradesthecapsularpoly-saccharideofE.
coliK5,thusallowingthephageaccesstooutermembranereceptors[34].
WhilstthepreciseidentityofthepolysaccharidetargetofFSH19Tsp2isatpresentunknown,itispossiblethatthistailspikeplaysamajorroleinhost-rangedetermination.
PlaqueassaysusingphagesVi01andFSH19againstapanelofS.
TyphimuriumandS.
TyphiBRD948indicatethatthesephagesarespecificfortheirhost(Table1).
ThisisperhapsnotsurprisingsinceVi01orf171c(Vi01Tsp2)possessesa9-0-acetylesterase/acetylxylanesterasedomain(residues343-445;DUF303)thattargetstheacetyl-modificationoftheS.
TyphiVicapsule[22].
ThepectatelyasedomainfoundinFSH19Tsp2ismorethanlikelyinvolvedinthedegradationofapolysacchar-idepresentonthesurfaceofS.
TyphimuriumU288.
Ananyetal.
[23]alsoreportthatSboM-AG3isShigella-specificandisunabletoformplaquesonlawnsofanyoftheSalmonella,Escherichia,andListeriastrainstested.
Aproteindomaincomposedofparallelright-handedb-helicesisalsofoundinFSH19Tsp3-theP22tailspikedomain.
ManytailspikeproteinspossessanN-terminalbindingdomaininvolvedinattachingthepro-teintothevirionhead/tailstructure(thesequenceofwhichisoftenhighlyconservedbetweenphageswithsimilarmorphologies),acentralcatalyticdomain(con-tainingthetrimericparallelright-handedb-helices),andaC-terminaltrimerizationdomain(involvedinstabiliza-tionofthetrimerictailspike).
Thetrimericb-helicesthatformthecatalyticdomainbindandcleavepolysac-charidespresentinbacteriallipopolysaccharide(LPS).
P22tailspike(Gp9)utilizesthisendo-rhamnosidaseactivitytodegradetheSalmonellaOantigen[35].
Clea-vageofa-L-rhamnose(1-3)a-D-galactosefoundinthetrisaccharide-repeatsofS.
TyphimuriumLPStakesplaceontheexternalsurfaceofthetailspike.
Thestructureandarrangementoftheparallelb-helixtailspikedomainsresultinasolvent-exposedexteriorthatfea-turestheproposedcatalyticresiduesGlu-359,Asp-392,andAsp-395[27,36].
TheactivesiteresiduesandtheirsequenceenvironmentsareconservedinFSH19Tsp3,andthereforecanbepostulatedtooperateinamannerakintotheP22tailspikeanditsrelatives.
Thus,thereisapossibilitythatFSH19Tsp3actsintwodistinctways.
Firstly,FSH19Tsp3mayfunctionasaninitialsteptowardsirreversibleadsorptionofthephageparticletoitshost,hydrolyzingtheouterS.
TyphimuriumLPSlayerinordertoprovideaccesstoapreviouslyinacces-sibleoutermembranereceptor.
Secondly,theendo-rhamnosidaseactivityassociatedwithP22-liketailspikesmayaidphageprogenyinfreeingthemselvesfromcellu-lardebrisencounteredduringhostlysis[37].
Tsp3ofFSH19hasanN-terminalsequencesimilartoVi01orf172c(Vi01Tsp3),SboM-AG3orf00207(SboM-AG3Tsp1)andDet7tailspike.
ThesephagesareHootonetal.
VirologyJournal2011,8:498http://www.
virologyj.
com/content/8/1/498Page10of14allMyoviridaeofsimilarmorphologies.
Thereforetherequirementsofhavingtoattachthetailspiketosimilarbaseplatestructuresmorethanlikelyplacesconstraintsonsequencedivergence.
Likewise,thealignmentsforthePodoviridaetailspikesshowN-terminalconserva-tionbetweenrelatedphage,followedbyconservedcata-lyticandC-terminaldomainsthataresharedbetweenMyoviridae,PodoviridaeandSiphoviridae.
Interestingly,P22,ST64T,andST104asmembersofthePodoviridaeare98-99%identicalovertheir667aminoacidtailspikeproteins,andsimilarlySiphoviridaemembersSETP12andSETP13alsohavealmostidenticaltailspikesequences.
DespitethattheSETP12and13phagesequencesaremarkedlydifferentfromP22,ST64T,andST104overtheN-terminalregions;however,theybecomeconservedbetweenresidues140to684.
Asnotedabove,thereappearstobesequencedivergenceconstraintsplacedonconservedN-terminalregionsassociatedwiththemyovirusesFSH19,Vi01,SboM-AG3andDet7.
SimilarlyitappearstherearestructuralrestrictionsimposedonthePodoviridaeandSiphoviri-daeN-terminaltailspikesequences.
SimilaritiesbetweenFSH19Tsp3,Det7andtheP22-likesequencesbegintenaminoacidsaftertheboundarymotifG-G-V-G-L-G-A-WthatappearsinFSH19Tsp3andDet7(thismotifalsosignalsthedivergenceofthetailspikesequencesofphagesVi01andSboM-AG3fromFSH19andDet7).
FSH19Tsp3andDet7TspshowstrikingconservationwiththeP22-likesequencesoverthecatalyticdomain,uptoandincludingtheactivesiteresiduesGlu-359,Asp-392andAsp-395.
However,immediatelyaftertheactivesiteinFSH19Tsp3(residues440-698)sequenceconservationbreaksdown.
ForP22-liketailspikeproteins,theC-terminalresidues585-596formanintertwinedregionthatallowsextensivehydrogenbondingbetweensubunits,whilstresidues606-664formafive-strandedandathree-strandedb-sheetregion[27].
ClustalW2alignmentoftheseregionsshowthatallthealignedP22-liketailspikescontainconservedresiduesattheselocations(includingtheMyovirusDet7),thatsetthemapartfromFSH19Tsp3(Figure4C).
Theeightaminoacidsspanningresidues585-592(V/G-G-P/A-G-S/T-G-S-A-W)retainasequencemotifsimilartothatfoundattheN-terminalboundaryofFSH19Tsp3,andthetail-associatedproteinsalignedinFigure4A.
However,thismotifisabsentattheC-terminalendofFSH19Tsp3.
P22-liketailspikemodulesmaywellhavedescendedfromacommonancestorwiththeflexibilitytodivergetheirproteinsequenceswhilstretainingthetopologyoftheright-handedparallelb-helices.
Ithasbeenproposedthatthisflexibilityhasallowedtheproteindomaintobecomewidespreadamongstbacteriophages[33],withthepossibilityoflaterdomaininterchangecreatingfurtherdiversity[38].
Insummary,wehavesequencedthegenomeofFSH19,apromisingcandidateforphagebiosanitizationofS.
Typhimuriuminthefoodproductionenvironment.
The157,785bpcirculardsDNAgenomewasfoundtocontainnogenesassociatedwithtoxicityorlysogeny,whichisapre-requisiteforsuchapplications.
AnalysisofthenucleotidesequenceofFSH19revealedonlytwocloserelativesinthedatabase:S.
Typhi-specificVi01andShigella-specificSboM-AG3thatcollectivelyformtheVi01-likephagefamily.
Majordifferenceswereevi-dentbetweenFSH19andVi01inthreedifferenttailspikeproteins.
TwotailspikesfromFSH19containproteindomainsassociatedwiththedegradationofpolysaccharidescommontoSalmonellaLPS,namelythepectatelyase(Tsp2)andP22tailspike-like(Tsp3)familiesofproteins.
TheacquisitionofthesedomainsisthemostobviousreasontoexplainthedifferenthostspecificitiesofFSH19,Vi01(seeminglyhost-restrictedduetothepresenceofaViantigen-degradingtailspikeproteindomain)andSboM-AG3(whosetailspikesareasyetundefined).
Basedonaminoacidsequenceanaly-sisthetailspikesfromallthreephagesappeartoformright-handedparallelb-helicalstructures.
Thisappearstobeanevolutionarily-conservedstructureforallthreetailspikesfoundintheVi01-likephage.
However,thisconservationiscoupledtotheexchangeofindividualproteindomainswithinthesestructuresthatmayulti-matelydeterminehostrange.
MaterialsandmethodsIsolationofFSH19Forphageisolation,sewageeffluentwasfilteredthrough0.
2μmMinisartfilters(SartoriusBiotech.
Cat.
No.
16534)andthefiltratecollectedinsterileuniversalsandstoredat4°Cuntilrequired.
ForS.
TyphimuriumlawnpreparationNZCYMbroth(DifcoCat.
No.
240410)cul-tureswerepreparedandincubatedovernightat37°Cwithshaking.
Followingthis,theovernightculturewasusedtoseedfreshNZCYMbrothcontaining10mMMgSO4(SigmaAldrichChemicalsCat.
No.
M2643)whichwasthenincubatedfor2hoursat37°Cwithshaking.
Tomolten(temperedto~50°C)NZCYMtopagarcontaining0.
6%BacteriologicalAgarNo.
1(OxoidCatNo.
LP0011)500μLoftherequiredSalmonellawasadded,followedby500μLfiltrate,andthemixturewaspouredontoNZCYMagarplates.
Theplateswerelefttosetonthebenchfor20minutesbeforebeinginvertedthenincubatedovernightat37°C.
Anyplaquesidentifiedwerepickedusingsterilepipettetipsandresuspendedin500μLSMbuffer(50mMTris-HCl[pH7.
5],100mMNaCl,8mMMgSO4:7H2O,0.
01%gelatin,pH7.
5),incu-batedat37°Cfor1hour,thenserialdilutedinSMbuf-fer.
A25μLvolumeofeachdilutionwasthenaddedtoSalmonella/NZCYMtopagar,andlawnswerepreparedHootonetal.
VirologyJournal2011,8:498http://www.
virologyj.
com/content/8/1/498Page11of14asdescribedabove.
ThisprocesswasrepeatedthreetimesinordertoobtainasingleclonalisolateofFSH19.
PhagehostrangeassaysTheabilityofphagesFSH19andVi01tolysevariousS.
TyphimuriumserovarsandS.
TyphiBRD948(kindlyprovidedbyD.
PickardWellcomeTrustSangerCentre,UK)wasdeterminedasfollows.
S.
TyphimuriumandS.
TyphiBRD948topagarlawnswerepreparedasdescribedabove,withtheexceptionofthelatterbeinggrownonsupplementedminimalmediaasdescribedbyTacketetal.
[39].
Toeachbacteriallawntriplicate20μLvolumesoflog107PFUmL-1dilutionsoftherequiredphagewereapplied.
Followingasufficientdryingperiod,plateswereinvertedandincubatedovernightat37°C.
Thefollowingday,eachplatewasobservedforlysisandtheresultsareshowninTable1.
TransmissionelectronmicroscopyAfreshly-preparedhightitrephagesuspensionofFSH19wassedimentedat34,900*gfor2hours(4°C).
Followingcentrifugation,thesupernatantwasdecantedandeachphagepelletwaswashedtwicewith0.
1Mammoniumacetatefor1hourat25,000*g.
Thewashsolutionwasdiscardedand2mLSMbufferaddedtoeachcentrifugetube.
Phagepelletswererecoveredfol-lowingovernightincubationat4°Cwithgentleshaking.
Asmalldropofwashedphagesuspensionwasspottedontoacarbon-coatedcoppermeshgridandallowedtositfor3minutes.
Excessphagesuspensionwasthenremovedwithfilterpaper.
Fornegativestainingonedropofphosphotungsticacid[pH7.
4]wasaddedtoeachgrid,andexcessstainwasremovedoneminutelaterwithfilterpaper.
Eachgridwasthencoveredandallowedtodryfor15minutes.
ImagesweretakenwithaFeiTecnaiBiotwinTEM(FeiCompany,USA).
FSH19DNAextractionandsequencingAhightitresuspensionofFSH19(~1010PFUmL-1)wassubjectedtoasingle-stepcaesiumchloride(CsCl)purificationprocedureasfollows.
CsCl(MelfordsCat.
No.
MB1006)wasaddedtothehightitrephagesuspen-siontoafinalconcentrationof0.
75gmL-1.
Sampleswerethensubjectedtoultracentrifugationat264,000*gfor24hoursinaBeckmanTL100ultracentrifuge(15°C).
Extractionofthebandcontainingpurifiedhighly-concentratedFSH19wasthenperformedwithasterile20-gaugehypodermicneedle,andthebandwassubse-quentlycollectedinasteriletube.
ResidualCsClwasremovedfromthesampleusinganAmiconUltra-0.
530kDaMWCOcentrifugalfilterdevice(MilliporeCat.
No.
UFC503008)asfollows.
Briefly,phage-CsClsolu-tionwasaddedtothecolumn,whichwasthenspunat17,900*ginabenchtopcentrifugefor30mins.
ThecolumnwasthenwashedtwicewithSMbufferat17,900*gfor2minutes.
Toelutetheretainedphage,SMbufferwasaddedtothecolumnwhichwastheninvertedandplacedinafreshcollectiontube.
Thecol-umnwasthencentrifugedat17,900*gfor10minutestorecoverthephage.
FSH19DNAwasisolatedfromthepurifiedstockusingthephenol-chloroformextrac-tionmethodwithslightmodificationsasfollows.
Anequalvolumeof10mgmL-1ProteinaseK(FisherScien-tificCatNo.
BPE1700-500)wasaddedtotheCsCl-pur-ifiedsample,followedbydetergentsolution(10ngmL-1ProteinaseKin50mMEDTA,50mMTris-HCl[pH8],1%N-lauroylsarcosine(SigmaAldrichChemicals,CatNo.
L9150)).
Thesolutionwasthenincubatedover-nightat55°C.
Followingthis,phageDNAwasextractedusingphenol:chloroformandethanolprecipitation[5].
TheextractedFSH19DNAwaspurifiedusingaDNAwizardpurificationkit(Promega,CatNo.
A1120)thensubjectedtowholegenomeamplificationusingaRepli-Gkit(Qiagen,UKCat.
No.
150023).
GenomicDNAwasfragmentedto500bpusingaCovarisS2sonicator(CovarisInc.
,USA)andlibrariesconstructedusingaNEBNextDNASamplePrepMasterMixSet2(NewEnglandBiolabsCat.
No.
E6070S).
ThelibrariesweresubsequentlysequencedusingtheRoche454GSFLXsystem(RocheDiagnostics,USA).
ThefullysequencedFSH19genomewasannotatedusingacombinationofCLCGenomicsWorkbench(CLCBio,Denmark)andArtemissoftware[40].
Forcompari-sonsbetweenthegenomesofFSH19,Vi01,andSboM-AG3,ACTsoftwarewasused[41],andcomparisonfilesweregeneratedusingtheweb-basedprogrammeDouble-ACThttp://www.
hpa-bioinfotools.
org.
uk/pise/double_-act.
html.
NucleotideandproteinsearcheswereperformedusingtheBlastsearchalgorithm[42].
Fortheidentificationofproteindomainsofknownfunction,searchesofthePfamdatabaseweremade[43],whilstproteinalignmentsweregeneratedusingClustalW2[44].
Betawrapwasusedtoidentifypotentialright-handedpar-allelb-helicesinthetailspikeproteinsencodedbyFSH19,Vi01,andSboM-AG3[45].
FortheidentificationofphageregulatoryelementsPhireanalysisoftheFSH19genomewasperformed[46],andtRNAscan-SE1.
21wasusedtoidentifytRNAgenes[47].
ThecompleteannotatedFSH19genomesequencehasbeendepositedintheNCBIdatabase(GenbankAcc.
No.
JN126049).
Abbreviationsgp:geneproduct,usedinthecontextoffunctionalhomologuestocoliphageT4proteins.
AcknowledgementsSPTHistherecipientofaUKFoodStandardsAgencypost-graduateresearchaward.
Hootonetal.
VirologyJournal2011,8:498http://www.
virologyj.
com/content/8/1/498Page12of14Authordetails1DivisionofFoodSciences,SchoolofBiosciences,UniversityofNottingham,SuttonBoningtonCampus,LoughboroughLE125RDUK.
2DeepSeq,UniversityofNottingham,QueensMedicalCentre,NottinghamNG72UHUK.
Authors'contributionsSPTHandIFCcontributedtothewritingofthismanuscript.
SPTHcontributedtoalltheexperimentalworkreportedhere.
ARTcontributedtotheassemblyandDNAsequenceinterpretation.
JRandRWperformedDNAsequencing.
IFCwastheprincipalinvestigatorandprovidedallfacilitatestocompletethiswork.
Allauthorsapprovedthefinalmanuscript.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
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